1. Field of the Invention
The present invention relates to a novel expression vector which is capable of enhancing gene expression through gene amplification in mammalian cells. The expression vector is organized as an inverted dimer (ID) and comprises, in addition to the gene to be expressed, a nuclear anchoring element and at least one inverted repeat element, preferably two inverted repeat elements. The present invention further relates to a method for enhancing gene expression by transfecting the expression vector to a mammalian cell, preferably a human cell.
2. Description of the Prior Art
A conventional gene expression method is used to express human recombinant proteins in a prokaryotic expression system. Most human recombinant proteins require post- and/or peri-translational modifications such as glycosylation, g-carboxylation, or g-hydroxylation. Therefore, a well-known problem with prokaryotic expression systems is that prokaryotic expression systems do not carry out post- and/or peri-translational modifications as mammalian expression systems do. Proteins requiring post- and/or peri-translational modifications for normal function cannot be expressed properly with prokaryotic expression systems.
Another conventional gene expression method widely used to express said human recombinant proteins is a mammalian expression system. A mammalian expression system uses mammalian cells because of their ability to undergo post-translational modifications and peri-translational modifications.
In particular, a mammalian expression system using Chinese hamster ovary (CHO) cells has become a routine and convenient expression system for expressing biopharmaceutical proteins for therapeutic and diagnostic uses.
A conventional method for enhancing gene expression in mammalian cells comprises a step of amplifying a gene by a step-by-step selection against a concentration-increasing selection agent. An embodiment of the conventional method for enhancing gene expression in mammalian cells uses Chinese hamster ovary (CHO) cells and amplifies a dihydrofolate recuctase (DHFR) gene with a step-by-step selection against methotrexate (MTX). See Omasa, 2002, J. Biosci. Bioeng. 94: 600-605.
However, the step-by-step selection is tedious and time-consuming. The process takes months to complete with cells expressing genes in high levels, and the post-translational modifications in non-human mammalian cells are not identical to that in human cells. Moreover, such conventional gene expression enhancing method with gene amplification has not been widely employed in human cells in the production of biopharmaceutical proteins for therapeutic and diagnostic uses. In general, human proteins generated in human cells should have beneficial properties in comparison with their counterparts produced in non-human systems.
It has been observed that a mismatch repair (MMR) system in human cells strongly suppresses gene amplification. See Lin et al., 2001, Nucleic Acids Res 29, 3304-3310; Chen et al., 2001, Proc Natl Acad Sci USA 98, 13802-13807. Thus gene expression enhanced by gene amplification is strongly suppressed in MMR+ (MMR-normal/MMR-proficient) cells. HCT116 cells are MMR− (MMR-deficient) due to a mutation in the hMLH1 gene. It is shown that HCT116 cells allow gene expression enhancement by gene amplification. In contrast, HCT116+Ch3 (MMR+ due to introduction of chromosome 3 which carries a wild-type hMLH1 gene) or HCT116+hMLH1 (MMR+ due to introduction of a cDNA which carries a wild-type hMLH1 gene) suppresses a drug resistant gene expression enhanced by gene amplification.
To overcome the shortcomings of the conventional gene expression methods and the conventional method for enhancing gene expression in mammalian cells (especially in human cells), the present invention provides a method to mitigate or obviate the aforementioned problems.